Millions of people are taking aspirin as therapy to reduce the risk of heart attacks and other cardiovascular events. These include persons with elevated cholesterol, a family history of heart disease, or other risk factors for cardiovascular disease. Among those with risk factors, nearly all persons with implanted cardiovascular devices are at elevated risk of clot formation and embolization and are prescribed some anti-platelet agent, usually aspirin. In addition, many healthy people without a recognized elevated risk of cardiovascular disease also take aspirin as a precaution.
Platelets function to stop bleeding by forming clots, and they initiate the process of wound healing. This occurs when platelets are activated, causing them to change shape, adhere, spread, release chemical messengers and activators, aggregate, and assemble with fibrin.
But platelet activation and clot formation can also place a person at risk of pathological cardiovascular events. For example, venous blood clot formation in the legs, a condition known as deep vein thrombosis, creates the risk that the blood clots could embolize (break apart) and result in clot entrapment in the lungs or the brain, causing pulmonary embolisms and stroke-related conditions. Platelet activation and fibrin formation in other locations in some persons create aggregates and small clots in the arterial circulation that can also lead to embolization and strokes.
Each year, approximately 500,000 heart valves are implanted in the United States. Although biomaterial advancement has somewhat reduced the risk of thrombosis (clot formation), all patients with mechanical heart valves are at increased risk of clot formation, embolization, and stroke, and are usually placed on aspirin therapy.
Arterial stents are another type of device placed in the circulatory system that place patients at risk from platelet activation. Arterial stents are placed in clogged coronary and carotid arteries to provide oxygen to cardiac tissue. They are typically around 5 mm in diameter and are made from stainless steel or other materials. Due to the introduction of a foreign material in the blood stream, platelets can become activated and attach to the wall of the stented vessel. This leads to reocclusion (restenosis) of the stented vessel, which is a very significant risk in patients with arterial stents. Restenosis in the first 28 days is reported in 0.5 to 8% of persons receiving stents.
To reduce these and other risks of cardiovascular pathology, millions of patients are placed on anti-platelet drugs, most commonly aspirin.
It is useful here to briefly summarize the biochemical events of hemostasis (the cessation of bleeding) and aspirin's role in inhibiting the process. Normal intact vascular endothelium does not initiate or support platelet adhesion (although in certain vascular diseases platelets may adhere to intact endothelium). Vascular injury, however, exposes the endothelial surface and underlying collagen. Following vascular injury, platelets attach to adhesive proteins such as collagen via specific glycoproteins on the platelet surface. This adhesion is followed or accompanied by platelet activation, where platelets undergo a shape change from a disc shape to a spherical shape with extended pseudopodia. At this time, the platelet release reaction also occurs. The platelets release biologically active compounds stored in the cytoplasmic bodies that stimulate platelet activation or are otherwise involved in clotting reactions. These include ADP, serotonin, thromboxane A2, and von Willebrand factor.
Following activation, glycoprotein receptors on the surface of the platelets undergo a conformational change from a relatively inactive conformation to an activated form. The activated receptors mediate the adhesion of more platelets by adhering to the circulating plasma protein fibrinogen, which serves as a bridging ligand between platelets. The adhesion and aggregation of platelets constitutes primary hemostasis.
Secondary hemostasis stabilizes the platelet mass by forming a fibrin clot. The fibrin clot is the end product of a series of reactions involving plasma proteins. The process is known as blood coagulation. In coagulation, fibrin is formed from fibrinogen, a large circulating plasma protein, by specific proteolysis. In the process, the protein thrombin is consumed. Fibrin monomers next spontaneously associate to form polymers and form a loose reinforcement of the platelet plug. Fibrin polymers are then cross-linked by certain enzymes. The fibrin polymer also traps red cells and white cells to form a finished clot.
Platelets are activated by a variety of stimuli. Collagen, ADP, thrombin, and physical shear stress all activate platelets. One of the first steps in activation is that a platelet membrane phospholipase, phospholipase A2, cleaves membrane lipids to release the fatty acid arachidonic acid. Arachidonic acid is oxidized in the platelet by the enzyme cyclooxygenase to the prostaglandin PGG2. PGG2 can be enzymatically converted to PGH2, and PGH2 is converted by thromboxane synthetase to thromboxane A2 (TxA2).
TxA2 is a very potent activator of platelets and greatly amplifies the platelet release reaction, where the platelets secrete the contents of certain cytoplasmic bodies, including alpha granules and dense bodies. Among the components secreted from dense bodies are ADP, Ca++, Mg++, and serotonin.
Aspirin acts by acetylating and inactivating cyclooxygenase-1 in platelets, preventing the synthesis of TxA2. By preventing the synthesis of TxA2, aspirin significantly reduces platelet activation and thus reduces clotting. Aspirin inactivates cyclooxygenase-1 (COX1) at a lower dose and more completely than it inactivates or inhibits another isoform of cyclooxygenase, cyclooxygenase-2. COX1 is the predominant cyclooxygenase in platelets. COX2 is involved in inflammation. (Vane, J. R., et al., 2003, The mechanism of action of aspirin, Thrombosis Research 110: 255.)
Thus, by inhibiting platelet activation, aspirin for most patients is an effective agent to prevent clots and pathological cardiovascular events. But many people are resistant to aspirin. In one study, 5.5% or 9.5% of patients were resistant to aspirin, as assayed by two different techniques, and 23.8% of patients were semi-resistant (Gum, P. A., et al., 2001, Am. J Cardiology 88: 230). Other studies estimate 5-40% of patients are aspirin resistant, depending on the assay and the population studied (Bhatt, D. L., 2004, J. Am. College of Cardiology Vol. 43, No. 6, 2004). This is very important, because aspirin resistance is significantly associated with an increased risk of death, myocardial infarction, or cerebrovascular accident (Altman, R., et al., 2004, Thrombosis J. 2: 1).
It is important to identify patients resistant to aspirin or other COX1 inhibitors, because if they are identified they can be placed on other platelet inhibitors that act by a different mechanism. This is important not only for proper treatment of the patients, but also for cost savings. The other platelet inhibitors are much more expensive than aspirin, so it would be extremely expensive to indiscriminately prescribe them. (Other platelet inhibitors include ADP inhibitors such as ticlopidine, and monoclonal antibodies that block the GPIIbIIIa receptor such as RHEOPRO.) Physicians are only likely to prescribe them when it can be shown that aspirin is not working.
Various techniques have been used to measure platelet function and aspirin resistance. Among these are platelet aggregation. In this technique, platelet-rich plasma was prepared from whole blood. ADP and arachidonic acid were added to activate the platelets. And aggregation of the platelets was measured by optical density changes. (Gum, P. A., et al., 2001, Am. J Cardiology 88: 230.) Another technique uses a device named the platelet function analyzer-100 (PFA-100). The PFA-100 uses a disposable cartridge with an aperture cut into a collagen-coated membrane infused with either ADP or epinephrine. Whole blood (approximately 1 ml) is pumped through the aperture at high shear rate. The blood comes into contact with the membrane where platelets adhere and aggregate. A platelet plug forms, occluding the aperture and stopping blood flow. The closure time is a measure of platelet function. (Gum, P. A., et al., 2001, Am. J Cardiology 88: 230.) Another device used to measure aspirin response is the Accumetrix VERIFYNOW Aspirin Assay (www.accumetrics.com/products/ultegra_asa.html). This product uses a turbidity-based optical detection system. The device contains fibrinogen-coated beads, and a platelet agonist. Blood is withdrawn, citrated, and then mixed with the coated beads and the agonist. Aggregation of the platelets to the beads is measured optically.
Prior tests for aspirin response have various drawbacks. Many use significant volumes of blood. Some require time-consuming and labor-consuming processing of the blood. And some measure adhesion and aggregation of the platelets, which are complex phenomena that are the end result of several interacting steps, rather than more directly measuring steps more directly related to aspirin's inhibition of cyclooxygenase.
A new method of monitoring aspirin response or response to other COX1 inhibitors is needed. Preferably, the method would use a small volume of blood (e.g., less than a drop), use unprocessed whole blood, be fast, and be relatively specific for the pathway inhibited by aspirin, the COX1 pathway.